Identification of O-mannosylated Virulence Factors in <em>Ustilago maydis</em>

<div><p>The O-mannosyltransferase Pmt4 has emerged as crucial for fungal virulence in the animal pathogens <em>Candida albicans</em> or <em>Cryptococcus neoformans</em> as well as in the phytopathogenic fungus <em>Ustilago maydis</em>. Pmt4 O-mannosylates specific target proteins at the Endoplasmic Reticulum. Therefore a deficient O-mannosylation of these target proteins must be responsible for the loss of pathogenicity in <em>pmt4</em> mutants. Taking advantage of the characteristics described for Pmt4 substrates in <em>Saccharomyces cerevisiae</em>, we performed a proteome-wide bioinformatic approach to identify putative Pmt4 targets in the corn smut fungus <em>U. maydis</em> and validated Pmt4-mediated glycosylation of candidate proteins by electrophoretic mobility shift assays. We found that the signalling mucin Msb2, which regulates appressorium differentiation upstream of the pathogenicity-related MAP kinase cascade, is O-mannosylated by Pmt4. The epistatic relationship of <em>pmt4</em> and <em>msb2</em> showed that both are likely to act in the same pathway. Furthermore, constitutive activation of the MAP kinase cascade restored appressorium development in <em>pmt4</em> mutants, suggesting that during the initial phase of infection the failure to O-mannosylate Msb2 is responsible for the virulence defect of <em>pmt4</em> mutants. On the other hand we demonstrate that during later stages of pathogenic development Pmt4 affects virulence independently of Msb2, probably by modifying secreted effector proteins. Pit1, a protein required for fungal spreading inside the infected leaf, was also identified as a Pmt4 target. Thus, O-mannosylation of different target proteins affects various stages of pathogenic development in <em>U. maydis</em>.</p>